MEDICINE FOR TREATING CANCER BY ADMINISTERING A TOLL-LIKE RECEPTOR AGONIST AND LAG-3 IgG FUSION PROTEIN

ABSTRACT

The present invention provides a medicine comprising a Toll-like receptor agonist, LAG-3 protein, a variant or derivative thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 16/341,415, which is the U.S. National Stage application of PCT/JP2017/015227, filed Apr. 14, 2017, which claims priority from Japanese application no. JP 2016-200227, filed Oct. 11, 2016.

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 22, 2022, is named sequence.txt and is 6,658 bytes.

TECHNICAL FIELD

The present invention relates to a medicine or the like comprising a Toll-like receptor agonist and LAG-3 protein, a variant or derivative thereof.

BACKGROUND ART

Recently, cancer immunotherapies targeting cancer antigens expressed specifically in cancer cells have been developed. Of them, a “cancer vaccine therapy” is a method of inducing regression of cancer by administering a cancer antigen directly to patients to induce an immune response specific to a cancer antigen in the patient's body. As the cancer antigen to be administered to a patient, e.g., a cancer-antigen protein itself, a cancer-antigen derived peptide, a nucleic acid encoding them, a dendritic cell presenting a cancer antigen and a cancer cell itself, are used.

To enhance induction of an immune response by a cancer antigen, an adjuvant is administered together with the cancer antigen. As the adjuvant, e.g., cytokines stimulating various immunocompetent cells and Toll-like receptor (TLR) agonists are used.

As the Toll-like receptor agonist (TLR agonist) serving as an adjuvant, any one of TLR1 to TLR10 agonists can be used (for example, Non Patent Literatures 1 and 2). For example, TLR3, which recognizes virus-derived double-stranded RNA and accelerates production of type I interferon exerting a strong antiviral action, is a molecule of the innate immune system. A double-stranded RNA analogue, Poly I:C (also called as Polyinosinic:polycytidylic acid), which is known as a TLR3 agonist, is known to be used as a vaccine adjuvant (for example, Patent Literature 1). Also, TLR9, which recognizes a bacterium- or virus-derived unmethylated CpG DNA and exerts an action, is a molecule of the innate immune system. CpG ODN (synthetic nucleic acid CpG oligodeoxynucleotide) serving as a TLR9-ligand is known to have an adjuvant effect for vaccine.

It is known that LAG-3 is also used as an adjuvant for vaccine (for example, Patent Literature 2). LAG-3 is a posttranslational product of lymphocyte activation gene 3 and also called as CD223. LAG-3 binds to a MHC Class II molecule to negatively control proliferation of activated T cells and homeostasis maintenance of T cells, plays an important role in regulatory T cell (Treg) function and is also known to be involved in homeostatic regulation of plasmacytoid dendritic cells.

CITATION LIST Patent Literature

-   Patent Literature 1: National Publication of International Patent     Application No. 2011-506309 -   Patent Literature 2: National Publication of International Patent     Application No. 2001-510806

Non Patent Literature

-   Non Patent Literature 1: Oncolmmunology 1: 5, 699-716; August 2012 -   Non Patent Literature 2: Oncolmmunology 2: 8, e25238; August 2013

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a medicine comprising a novel combination of adjuvants.

Solution to Problem

The present inventors conducted various studies using various adjuvants singly or in combination. As a result, they found a novel combination of adjuvants serving as a medicine useful for, for example, inducing cancer immunity.

More specifically, the present invention is as follows.

[1]

A medicine comprising

a Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof.

[2]

The medicine according to [1] for combined administration of

the Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof.

[3]

The medicine according to [1] or [2], wherein the Toll-like receptor agonist is Toll-like receptor 3 agonist or a Toll-like receptor 9 agonist.

[4]

The medicine according to any one of [1] to [3], in which the Toll-like receptor agonist is Poly I:C or a salt thereof.

[5]

The medicine according to [4], wherein the Poly I: C is Poly ICLC.

[6]

The medicine according to any one of [1] to [5], wherein the LAG-3 protein, a variant or derivative thereof is a fusion protein of LAG-3 protein and IgG.

[6′]

The medicine according to any one of [1] to [6], further comprising a substance for inducing a specific immune response to at least one cancer cell.

[6″]

The medicine according to [6′], for combined administration of

the Toll-like receptor agonist,

LAG-3 protein, a variant or derivative thereof, and

the substance for inducing a specific immune response to at least one cancer cell.

[7]

The medicine according to any one of [1] to [6], further comprising at least one cancer-antigen derived peptide.

[8]

The medicine according to [7], wherein the at least one cancer-antigen derived peptide is an HSP70-derived peptide or a GPC3-derived peptide.

[9]

The medicine according to [7] or [8], comprising at least one cancer-antigen derived peptide selected from the group consisting of an HSP70-derived peptide and a GPC3-derived peptide,

Poly ICLC and

LAG-3 protein or a fusion protein of LAG-3 protein and IgG.

[10]

The medicine according to [9], comprising an HSP70-derived peptide having an amino acid sequence represented by SEQ. ID No: 7 or a GPC3-derived peptide having an amino acid sequence represented by SEQ. ID No: 16.

[11]

The medicine according to any one of [7] to [10], comprising at least two types of cancer-antigen derived peptides.

[12]

The medicine according to [11], comprising an HSP70-derived peptide and a cancer-antigen derived peptide except an HSP70-derived peptide.

[13]

The medicine according to [11], comprising a GPC3-derived peptide and a cancer-antigen derived peptide except a GPC3-derived peptide.

[14]

The medicine according to [11], comprising an HSP70-derived peptide and a GPC3-derived peptide.

[15]

The medicine according to [14], comprising an HSP70-derived peptide having an amino acid sequence represented by SEQ. ID No: 7 and a GPC3-derived peptide having an amino acid sequence represented by SEQ. ID No: 16.

[16]

The medicine according to [12], comprising an HSP70-derived peptide, a cancer-antigen derived peptide except an HSP70-derived peptide,

Poly ICLC and

LAG-3 protein or a fusion protein of LAG-3 protein and IgG.

[17]

The medicine according to [13], comprising

a GPC3-derived peptide, a cancer-antigen derived peptide except a GPC3-derived peptide,

Poly ICLC and

a LAG-3 protein or a fusion protein of LAG-3 protein and IgG.

[18]

The medicine according to [14], comprising

an HSP70-derived peptide, a GPC3-derived peptide,

Poly ICLC and

LAG-3 protein or a fusion protein of LAG-3 protein and IgG.

[19]

The medicine according to [18], comprising

an HSP70-derived peptide having an amino acid sequence represented by SEQ. ID No: 7 and a GPC3-derived peptide having an amino acid sequence represented by SEQ. ID No: 16.

[20]

The medicine according to any one of [1] to [19], for use in a cancer vaccine therapy.

[21]

The medicine according to any one of [1] to [20], wherein the medicine is an anti-cancer agent.

[22]

An adjuvant comprising

a Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof, for use in inducing a specific immune response to a cancer cell or for use in a cancer vaccine therapy.

[23]

A combination comprising

a Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof, for use in inducing a specific immune response to a cancer cell or in a cancer vaccine therapy.

[24]

A method for treating or preventing a disease in a patient, comprising administering

a Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof, to a patient in need thereof.

[25]

A method for inducing a specific immune response to a cancer cell, comprising administering

a Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof, to a patient in need thereof.

Advantageous Effects of Invention

According to the present invention, it is possible to provide a medicine comprising a new combination of adjuvants.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic protocol of an experiment for comparing effects of adjuvants in a cancer vaccine using a cancer-antigen derived peptide.

FIG. 2 is a graph showing changes in tumor size of Group 1, in which PBS was used as a control in the experiment of FIG. 1.

FIG. 3 is a graph showing changes in tumor size in Group 2, in which IFA was used as an adjuvant in the experiment of FIG. 1.

FIG. 4 is a graph showing changes in tumor size in Group 3, in which PT was used as an adjuvant in the experiment of FIG. 1.

FIG. 5 is a graph showing changes in tumor size in Group 4, in which Poly I:C was used as an adjuvant in the experiment of FIG. 1.

FIG. 6 is a graph showing changes in tumor size in Group 5, in which a combination of Poly I:C and CpG was used as an adjuvant in the experiment of FIG. 1.

FIG. 7 is a graph showing change in tumor size in Group 6, in which LAG-3Ig was used as an adjuvant in the experiment of FIG. 1.

FIG. 8 is a graph showing changes in tumor size in Group 7, in which a combination of LAG-3Ig and Poly I:C was used as an adjuvant in the experiment of FIG. 1.

FIG. 9A shows images of a tumor tissue stained with hematoxylin-eosin in the same experiment as in FIG. 1.

FIG. 9B shows images of cell nuclei and immune cells of a tumor tissue stained with a fluorescent dye in the same experiment as in FIG. 1.

FIG. 10 is a graph showing the measurement results (increase) of tumor size after tumor cells were again inoculated to a mouse in which an increase in tumor size was once successfully suppressed by a cancer vaccine (LAG-3Ig and Poly I:C were used as adjuvants) in the same experiment as in FIG. 1.

FIG. 11A is a graph showing the measurement results of proliferative ability of immune cells, which were prepared by taking a lymph node from a mouse inoculated with a cancer vaccine in the same experiment as in FIG. 1, separating the immune cells from the lymph node, and which were co-culturing with tumor cells previously inactivated by radiation exposure.

FIG. 11B shows graphs showing the measurement results of cytokines in the cell supernatant of the same co-culture as in the experiment in FIG. 11A.

FIG. 12A shows the measurement results of expression of cell surface marker molecules, PD-1, BTLA, TIGIT and LAG-3, present on the surface of CD8 positive immune cells, which were prepared by taking a lymph node from a mouse inoculated with a cancer vaccine in the same experiment as in FIG. 1 and separating the immune cells from the lymph node.

FIG. 12B shows the measurement results of expression of cell surface marker molecules, PD-1, BTLA, TIGIT and LAG-3, present on the surface of CD4 positive immune cells, which were prepared by taking a lymph node from a mouse inoculated with a cancer vaccine in the same experiment as in FIG. 1 and separating the immune cells from the lymph node.

FIG. 13A is a graph showing measurement results of proliferative ability of immune cells, and FIG. 13B is a graph showing measurement results of IFN-γ production.

FIG. 14 is a graph showing changes in tumor size of Group 1, in which PBS was used as a control.

FIG. 15 is a graph showing changes in tumor size of Group 2, in which Poly I:C was used as an adjuvant.

FIG. 16 is a graph showing changes in tumor size in Group 3, in which a combination of LAG-3Ig and Poly I:C was used as an adjuvant.

FIG. 17 is a graph showing changes in tumor size in Group 4, in which a combination of LAG-3Ig and Poly I:C, alone was used without using PIA peptide.

FIG. 18 is a graph showing changes in tumor size of Group 5, in which RIBOXXOL was used as an adjuvant.

FIG. 19 is a graph showing changes in tumor size of Group 6, in which a combination of LAG-3Ig and RIBOXXOL was used as an adjuvant.

FIG. 20 is a graph showing changes in tumor size of Group 7, in which a combination of LAG-3Ig and MPL was used as an adjuvant.

FIG. 21 is a graph showing changes in tumor size of Group 8, in which a combination of LAG-3Ig and Imiquimod was used as an adjuvant.

FIG. 22 is a graph showing changes in tumor size of Group 9, in which a combination of LAG-3Ig and CpG was used as an adjuvant.

FIG. 23 is a graph showing changes in tumor size of Group 1, in which IFA was used as an adjuvant.

FIG. 24 is a graph showing changes in tumor size of Group 2, in which Poly I:C was used as an adjuvant.

FIG. 25 is a graph showing changes in tumor size of Group 3, in which LAG-3Ig was used as an adjuvant.

FIG. 26 is a graph showing changes in tumor size in Group 4, in which a combination of LAG-3Ig and Poly I:C was used as an adjuvant.

FIG. 27A is a graph showing IFN-γ productions from immune cells isolated from patient's blood and cultured after an HSP70-derived peptide, a GPC3-derived peptide and a combination of Poly ICLC (product name: “Hiltonol”) and LAG-3Ig (IMP321) were administered 10 times to esophageal cancer patients.

FIG. 27B is a graph showing IFN-γ productions from immune cells isolated from patient's blood and cultured after an HSP70-derived peptide, a GPC3-derived peptide and a combination of Poly ICLC (product name: “Hiltonol”) and LAG-3Ig (IMP321) were administered 10 times to liver cancer patients.

FIG. 28A is a graph showing IFN-γ productions from immune cells isolated from patient's blood and cultured after an HSP70-derived peptide, a GPC3-derived peptide and a combination of Poly ICLC (product name: “Hiltonol”) and LAG-3Ig (IMP321) were administered 4 times to liver cancer patients.

FIG. 28B is a graph showing IFN-γ productions from immune cells isolated from patient's blood and cultured after an HSP70-derived peptide, a GPC3-derived peptide and a combination of Poly ICLC (product name: “Hiltonol”) and LAG-3Ig (IMP321) were administered 8 times to rectal cancer patients.

FIG. 28C is a graph showing IFN-γ productions from immune cells isolated from patient's blood and cultured after an HSP70-derived peptide, a GPC3-derived peptide and a combination of Poly ICLC (product name: “Hiltonol”) and LAG-3Ig (IMP321) were administered 8 times to esophageal cancer patients.

DESCRIPTION OF EMBODIMENTS

The present invention will be more specifically described below by way of embodiments; however, the present invention is not limited to the embodiments and can be modified in various ways and carried out.

A medicine according to the present invention includes a Toll-like receptor agonist (TLR agonist) and LAG-3 protein, a variant or derivative thereof.

In the specification, the “Toll-like receptor agonist (TLR agonist)” refers to a molecule, which binds to any one of Toll-like receptors and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR. As the TLR agonist, TLR agonists described in Non Patent Literatures 1 and 2, Trends in Immunology, Vol. 30, No. 1, 23-32, Immunity 33, Oct. 29, 2010, 492-503, World Journal of Vaccines, 2011, 1, 33-78 and Oncolmmunology 1:6, 894-907; September 2012, are known. These TLRs known in the art each can be used as the TLR agonist in the present invention.

As the TLR agonist, TLR1 to TLR10 agonists are mentioned, for example, a TLR3 agonist, TLR4 agonist, TLR7 agonist, TLR8 agonist, TLR9 agonist or TLR10 agonist can be used. Of them, a TLR3 agonist or a TLR9 agonist may be used as the TLR agonist.

In the specification, the “TLR1 agonist” refers to a molecule which binds to TLR1 and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR1. TLR1 recognizes e.g., a lipoprotein, to activate the innate immune system.

Examples of the TLR1 agonist include a lipoprotein, Triacylated lipopeptides and Zymosan.

In the specification, the “TLR2 agonist” refers to a molecule which binds to TLR2 and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR2. TLR2 recognizes, e.g., Acylated lipopeptides, to activate the innate immune system.

Examples of the TLR2 agonist include SMP-105, Acylated lipopeptides, Amphotericin B, Atypical LPS, Byglican, Forins, Glyco(phospho)lipids, Lipoteichoic acid, Peptidoglycan, Phenol soluble modulin and Zymosan.

In the specification, the “TLR3 agonist” refers to a molecule which binds to TLR3 and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR3. TLR3 recognizes a virus-derived double stranded RNA to activate the innate immune system. As the TLR3 agonist, a synthetic double stranded polynucleotide, Poly I:C, having an analogous structure to double stranded RNA is known; however, the TLR3 agonist is not limited to this.

A salt of Poly I:C may be used in the present invention; for example, a sodium salt thereof can be used. As the Poly I:C, Poly ICLC can be used. As the TLR3 agonist, RIBOXXOL can be used.

Examples of the TLR3 agonist include Poly I:C, Poly ICLC (product name: “Hiltonol”), RIBOXXOL, Ampligen, IPH-3102, cM362-139 and cM362-140.

As the TLR3 agonist, preferably a Poly I:C is used. Of the Poly I:Cs, Poly ICLC (product name: “Hiltonol”) stabilized with Poly-Lysine and Carboxymethylcellulose, is used.

In the specification, the “TLR4 agonist” refers to a molecule which binds to TLR4 and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR4. TLR4 recognizes a bacterium-derived lipopolysaccharide (LPS) to activate the innate immune system. As the TLR4 agonist, MPL is known; however, the TLR4 agonist is not limited to this. A salt of MPL may be used in the present invention; for example, a sodium salt thereof can be used.

Examples of the TLR4 agonist include MPL, MPLA, OM-174 (CRX-527), Picibanil (OK-432) and ONO-4007.

In the specification, the “TLR5 agonist” refers to a molecule which binds to TLR5 and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR5. TLR5 recognizes a bacterium-derived Flagellin to activate the innate immune system. As the TLR5 agonist, Flagellin (proteins) derived from various bacteria or recombinant Flagellin proteins are known; however, the TLR5 agonist is not limited to these.

Examples of the TLR5 agonist include CBLB502.

In the specification, the “TLR6 agonist” refers to a molecule which binds to TLR6 and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR6. TLR6 recognizes, e.g., a lipoprotein and activate the innate immune system.

Examples of the TLR6 agonist include Diacylated lipopeptides, Lipoproteins, Lipoteichoic acid and Zymosan.

In the specification, the “TLR7 agonist” refers to a molecule which binds to TLR7 and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR7.

Also, in the specification, the “TLR8 agonist” refers to a molecule which binds to TLR8 and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR8.

TLR7 and TLR8 recognize virus-derived single stranded RNA to activate the innate immune system. As the TLR7/8 agonist, Imiquimod is known; however, the TLR7/8 agonist is not limited to this. A salt of Imiquimod may be used in the present invention; for example, a sodium salt thereof can be used.

Examples of the TLR7/8 agonist include Imiquimod (S-26308, R-837), Resimiquimod (R-848), 3M-052, 852A, VTX-1463, VTX-2337, AZD8848 (DSP-3025), ANA773 and TMX-101.

In the specification, the “TLR9 agonist” refers to a molecule which binds to TLR9 and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR9. TLR9 recognizes bacterium- and virus-derived CpG DNA to activate the innate immune system. As the TLR9 agonist, CpG ODN is known; however, the TLR9 agonist is not limited to this. A salt of CpG ODN (CpG Oligodeoxynucleotide) may be used in the present invention; for example, a sodium salt thereof can be used.

Examples of the TLR9 agonist include CpG ODN such as CpG, CpG-28, CpG-685 (GNKG168), CpG-1826, CpG-7909 (PF-3512676, Agatolimod, promune (registered trademark)), ODN1585, IMO-2125, IMO-2055 (EMD1201081), ISS1018, MGN-1703, MGN-1706, AVE0675, QAX-935, SAR-21609, SD-101 and DIMS0150.

In the specification, the “TLR10 agonist” refers to a molecule which binds to TLR10 and provides the same stimulus as the stimulus provided by a natural ligand bound to TLR10. TLR10 recognizes, e.g., an acylated lipopeptide to activate the innate immune system.

Examples of the TLR10 agonist include an acylated lipopeptide.

In the specification “LAG-3 protein, a variant or derivative thereof” refers to LAG-3 protein, a functional variant or derivative thereof. The LAG-3 protein to be used in the present invention may be derived from any animal, for example, the protein can be derived from the same animal as the subject to which a medicine according to the present invention is to be administered. More specifically, if a medicine according to the present invention is administered to a human, human LAG-3 can be used. Human LAG-3 protein is a protein having an amino acid sequence represented by NCBI Accession No. P18627.5.

Examples of the functional variant of LAG-3 protein include, (i) an LAG-3 variant consisting of an amino acid sequence, which is the amino acid sequence of LAG-3 having addition, substitution, or deletion of one or several amino acids and having a function of LAG-3 protein required for exerting the effects of the present invention, (ii) an LAG-3 variant having a sequence identity with the amino acid sequence of LAG-3 of at least 80% or more, or 85% or more, 90% or more, 95% or more, 98% or more or 99% or more and having a function of LAG-3 protein required for exerting the effects of the present invention, and (iii) a partial polypeptide of LAG-3 protein, a variant defined in the above (i) or a variant defined in the above (ii) having a function of LAG-3 protein required for exerting the effects of the present invention.

In the specification, the “one or several amino acids” refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

In the specification, the “amino acid” is used in the broadest sense thereof and includes natural amino acids and artificial amino acid variants and derivatives. In the specification, examples of the amino acid include natural protein L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and derivatives; natural non-protein amino acids such as norleucine, β-alanine and ornithine; and chemically synthesized compounds having amino acid characteristics known in the art. Examples of non-natural amino acids include α-methyl amino acid (e.g., α-methylalanine), D-amino acid, histidine-like amino acid (e.g., β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine and α-methyl-histidine), amino acids with extra methylene in a side chain (“homo” amino acids) and amino acids (e.g., cysteine acid) in which the carboxylic acid functional group amino acid in a side chain is substituted with a sulfonic acid group.

Examples of the functional derivative of LAG-3 protein include a fusion protein of all or part of LAG-3 protein and another protein or a polypeptide, and LAG-3 protein attached with a sugar chain and/or a lipid. As an example of the functional derivative of LAG-3 protein, a fusion protein of LAG-3 and IgG (LAG-3Ig). Examples of the functional derivative of LAG-3 protein include the derivatives described in Journal of Translational Medicine 2014, 12: 97.

As the functional derivative of LAG-3 protein, for example, a fusion protein of LAG-3 and IgG, i.e., IMP321 (LAG-3Ig), can be preferably used.

A medicine according to the present invention may contain a TLR agonist and LAG-3 protein, a variant or derivative thereof. As the TLR agonist, a TLR3 agonist or a TLR9 agonist can be preferably used and a TLR3 agonist can be more preferably used. In a medicine according to the present invention, human LAG-3 and LAG-3Ig can be preferably used as the LAG-3 protein, a variant or derivative thereof.

As the combination of a TLR agonist and LAG-3 protein, a variant or derivative thereof, a combination of a TLR3 agonist or a TLR9 agonist, and LAG-3 protein, a variant or derivative thereof or a combination of a TLR agonist and LAG-3 protein or a derivative of LAG-3 protein is preferable; a combination of a TLR3 agonist or a TLR9 agonist and LAG-3 protein or a derivative of LAG-3 protein is more preferable; a combination of a TLR3 agonist and LAG-3 protein or a derivative of LAG-3 protein is further preferable; and a combination of a TLR3 agonist and a derivative of LAG-3 protein is further more preferable.

In the combination of a TLR agonist and LAG-3 protein, a variant or derivative thereof, Poly I:C or a salt thereof can be used as the TLR3 agonist, and Poly ICLC may be used as the Poly I:C. As the TLR3 agonist, e.g., Poly I:C, Poly ICLC (product name: “Hiltonol”), RIBOXXOL, Ampligen, IPH-3102, cM362-139 and cM362-140 may be used. As the TLR3 agonist, Poly ICLC (product name: “Hiltonol”) can be preferably used.

In the combination of a TLR agonist and LAG-3 protein, a variant or derivative thereof, CpG ODN or a salt thereof may be used as a TLR9 agonist and a sodium salt of CpG ODN may be used. As the TLR9 agonist, e.g., CpG, CpG-28, CpG-685 (GNKG168), CpG-1826, CpG-7909 (PF-3512676, Agatolimod, promune (registered trade mark)), ODN1585, IMO-2125, IMO-2055 (EMD1201081), ISS1018, MGN-1703, MGN-1706, AVE0675, QAX-935, SAR-21609, SD-101 and DIMS0150, may be used.

In the combination of a TLR agonist and LAG-3 protein, a variant or derivative thereof, as the LAG-3 protein, a variant or derivative thereof, the LAG-3 protein as mentioned above and a functional mutant or a functional derivative thereof can be used; a functional derivative of LAG-3 protein may be used; and a human LAG-3 and a fusion protein of LAG-3 and IgG (LAG-3Ig) may be used.

A medicine according to the present invention preferably contains a TLR agonist and LAG-3 protein, a variant or derivative thereof and further contains a substance for inducing a specific immune response to a cancer cell. Also, in the case of a medicine further comprising a substance for inducing a specific immune response to a cancer cell, the aforementioned combination of a TLR agonist and LAG-3 protein, a variant or derivative thereof can be used.

A medicine according to the present invention preferably contains a TLR3 agonist or a TLR9 agonist, LAG-3 protein or LAG-3 protein derivative, and a substance for inducing a specific immune response to a cancer cell. More preferably, a medicine according to the present invention contains a TLR3 agonist, LAG-3 protein or a derivative of LAG-3 protein, and a substance for inducing a specific immune response to a cancer cell. Further preferably, a medicine according to the present invention contains a TLR3 agonist, a derivative of LAG-3 protein, and a substance for inducing a specific immune response to a cancer cell.

If a medicine according to the present invention further contains a substance for inducing a specific immune response to a cancer cell, a cancer-antigen protein, a cancer-antigen derived peptide, nucleic acids encoding them, a cancer antigen-presenting cell and a tumor cell may be used as the substance for inducing a specific immune response to a cancer cell, and a cancer-antigen derived peptide is preferably used.

A medicine according to the present invention can be used for cancer immunotherapy by the following mechanism. The medicine, which is preferably administered by a subcutaneous or intradermal route, brings about the phenomena: (1) antigen presentation in the near dendritic cells, (2) activation of T cell priming in the near lymph node and (3) recognition and damage of a tumor at a tumor site by antigen peptide-specific CTLs induced.

Taking a case where a TLR3 agonist is used as the TLR agonist in the medicine according to the present invention, as an example, a substance for inducing a specific immune response to a cancer cell administered together with the TLR3 agonist is presented as an antigen and T cell priming is promoted.

Taking a case where LAG-3Ig is used as the LAG-3 protein, a variant or derivative thereof in the medicine according to the present invention, as an example, it is expected that antigen presentation to a substance for inducing a specific immune response to a cancer cell is promoted; at the same time, at a tumor site, an inhibitory signal against activated CTLs by, e.g., a tumor or a macrophage expressing a MHC class II molecule is blocked to suppress exhaustion of CTL.

It is considered that damage on a tumor is accelerated by the functions of the TLR3 agonist and LAG-3Ig independently of a substance for inducing a specific immune response to a cancer cell simultaneously administered.

In the specification, the “substance for inducing a specific immune response to a cancer cell” is not particularly limited as long as it is a substance capable of inducing an immune response necessary for breaking cancer cells in-vivo and inducing apoptosis of cancer cells; for example, a cancer-antigen protein, a cancer-antigen derived peptide, nucleic acids encoding them, a cancer antigen-presenting cell and a tumor cell itself are mentioned.

In the specification, the “cancer-antigen protein” is a protein specially expressed on a cancer cell and recognized/attacked by the immune system as a foreign body. The “cancer-antigen protein” may be expressed on cancer cells of all types and a specific type of cancer. As the cancer-antigen protein, a protein having strong immunogenicity and never be expressed in normal cells, is preferable. Examples of the cancer-antigen protein include, but are not particularly limited to, those described in Clin Cancer Res 2009; 15 (17) 5323. Specific examples thereof include, but are not particularly limited to, WT1, LMP2, HPVE6, HPVE7, EGFRv III, HER-2/neu, MAGE-A3, p53nonmutant, HSP70, GPC3, MUC1, Casp8, CDAM1, cMyb, EVC1, EVC2, Helios, Fas, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant, Proteinase3 (PR1), bcr-abl, Tyrosinase, Survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3, ALK, Androgen receptor, Cyclin B1, Polysialic acid, MYCN, PhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE A1, sLe, CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAXS, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, Legumain, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR, MAD-CT-2 and Fos-related antigen 1.

Suitable examples of the cancer-antigen protein include, but are not particularly limited to, HSP70, GPC3, MUC1 and gp100.

In the specification, the “cancer-antigen derived peptide” refers to a peptide having a part of the amino acid sequence of a cancer-antigen protein, or a peptide having a sequence, which is the same amino acid sequence as above and having addition, substitution or deletion of 1 or 2 amino acids, or a peptide having a sequence identity with the above amino acid sequence of 90% or more, 95% or more, 98% or more, or 99% or more and inducing immune which attacks cancer cells. The cancer-antigen derived peptide may consist of amino acid residues of 8 or more and 11 or less. Such a peptide, if it is subcutaneously administered to a patient, is incorporated into antigen-presenting cells, such as dendritic cells and macrophages, and presented on the surface of the cells together with HLA molecules. Precursor cells of cytotoxic T cell (CTL) having a reactivity to the peptide presented are clonally proliferated and mature CTLs that proliferated and differentiated migrate through the lymph flow into a cancer tissue. The mature CTLs attack a cancer cell on which a peptide having the same sequence as the peptide administered is expressed to induce apoptosis.

Examples of the HSP70-derived peptide as the cancer-antigen derived peptide include, but are not particularly limited to, peptides described in International Publication No. WO2016/056596. More specifically, a peptide comprising continuous 8 or more amino acid residues in the amino acid sequence represented by any one of SEQ. ID Nos: 1 to 15 and consisting of 11 or less amino acid residues, is mentioned.

In the present invention, if an HSP70-derived peptide is used, although it is not particularly limited, an HSP70-derived peptide having the amino acid sequence represented by any one of SEQ. ID Nos: 1 to 15 can be preferably used, and an HSP70-derived peptide having the amino acid sequence represented by the SEQ. ID No: 7 is more preferably used.

Examples of the GPC3-derived peptide as the cancer-antigen derived peptide include, but are not particularly limited to, peptides described in International Publication No. WO2016/143816. More specifically, a peptide comprising continuous 8 or more amino acid residues in the amino acid sequence represented by any one of SEQ. ID Nos: 16 to 26 and consisting of 11 or less amino acid residues, is mentioned.

In the present invention, if a GPC3-derived peptide is used, although it is not particularly limited, a GPC3-derived peptide having the amino acid sequence represented by any one of SEQ. ID Nos: 16 to 26 can be preferably used, a GPC3-derived peptide having the amino acid sequence represented by the SEQ. ID No: 16 is more preferably used.

Examples of the MUC1 derived peptide as the cancer-antigen derived peptide, include, but are not particularly limited to, peptides described in International Publication No. WO2016/143814. More specifically, a peptide comprising continuous 8 or more amino acid residues in the amino acid sequence represented by any one of SEQ. ID Nos: 27 to 39 and consisting of 11 or less amino acid residues, is mentioned.

These antigen-derived peptides are disclosed just as examples. The “substance for inducing a specific immune response to a cancer cell” used in the medicine according to the present invention is not limited to these antigen-derived peptides.

In the specification, the “nucleic acid” is not particularly limited as long as it is a nucleic acid encoding a cancer-antigen protein or a cancer-antigen derived peptide, and includes RNA, DNA, PNA, LNA or a chimeras of two or more of these. These nucleic acids can be inserted into, e.g., a vector, in accordance with a known method, and then administered to patients and express a cancer-antigen protein or a cancer-antigen derived peptide, in vivo.

In the specification, the “cancer antigen-presenting cell” refers to an antigen-presenting cell presenting a cancer-antigen derived peptide on the surface thereof via binding to an HLA molecule. As the antigen-presenting cell, dendritic cells and macrophages can be used. The dendritic cells have particularly high CTL inducibility. The antigen-presenting dendritic cells can be obtained, for example, by separating mononuclear cells from the patient's own peripheral blood, differentiating the mononuclear cells into immature dendritic cells and thereafter further differentiating mature dendritic cells by adding a cancer-antigen protein or a cancer-antigen derived peptide to a medium.

As a cancer vaccine presently under development, vaccines using dendritic cells sensitized with a tumor cell extract, dendritic cells sensitized with a cancer antigen/GM-CSF fusion protein and a combination of noninfectious virus-like particles of a HPV-derived L1 protein and an adjuvant, are known. These are included in the “substance for inducing a specific immune response to a cancer cell”.

A medicine according to the present invention can contain at least one “substance for inducing a specific immune response to a cancer cell”; and may contain at least two types of “substances for inducing a specific immune response to a cancer cells”. For example, a medicine according to the present invention may contain, as “the substance for inducing a specific immune response to a cancer cell”, a single cancer-antigen derived peptide, which is derived from a single type of cancer-antigen protein or may contain cancer-antigen derived peptides, which are derived from at least two types of cancer-antigen proteins.

A medicine according to the present invention preferably contains at least one cancer-antigen derived peptide.

As the at least one type of cancer-antigen derived peptide, which is not particularly limited, a single type of cancer-antigen derived peptide may be used or at least two types of cancer-antigen derived peptides may be used.

As the cancer-antigen protein in the cancer-antigen derived peptide, which is derived from a cancer-antigen protein, a protein selected from the cancer-antigen proteins listed in the “cancer-antigen protein”, can be mentioned.

As the at least two types of cancer-antigen derived peptides, at least two types of cancer-antigen derived peptides derived from different cancer-antigen proteins may be used, or at least two types of different cancer-antigen derived peptides derived from the same cancer-antigen protein, may be used.

As the at least two types of different cancer-antigen derived peptides, at least two types of different proteins selected from the cancer-antigen proteins listed in the “cancer-antigen protein” can be mentioned.

More specifically, as the cancer-antigen derived peptide, HSP70, GPC3, MUC1 or gp100-derived peptide is preferably used and an HSP70-derived peptide or a GPC3-derived peptide is more preferably used.

If a medicine according to the present invention contains a single type of cancer-antigen derived peptide, it is preferable to use an HSP70-derived peptide or a GPC3-derived peptide.

If a medicine according to the present invention contains at least two types of cancer-antigen derived peptides, it is preferable to use an HSP70, GPC3, MUC1 or gp100-derived peptide as one of the at least two types of cancer-antigen derived peptides.

As the two types of cancer-antigen derived peptides, a combination of an HSP70-derived peptide and a cancer-antigen derived peptide except an HSP70-derived peptide, a combination of a GPC3-derived peptide and a cancer-antigen derived peptide except a GPC3-derived peptide, and a combination of an HSP70-derived peptide and a GPC3-derived peptide, are preferably used.

As the cancer-antigen derived peptide except an HSP70-derived peptide and the cancer-antigen derived peptide except a GPC3-derived peptide, cancer-antigen derived peptides, which are derived from antigen proteins, which are selected from the cancer-antigen proteins listed in the “cancer-antigen protein” and, from which HSP70 and GPC3 are respectively removed, are mentioned.

In the present invention, if two types of cancer-antigen derived peptides are used, it is preferable to use a combination of two types of peptides, i.e., an HSP70-derived peptide and a GPC3-derived peptide.

If a single type of cancer-antigen derived peptide is used, it is preferable to use an HSP70, GPC3, MUC1 or gp100-derived peptide and more preferable to use an HSP70-derived peptide or a GPC3-derived peptide.

At least two types of “substances for inducing a specific immune response to a cancer cell” may be at least two types of substances selected from cancer-antigen proteins, cancer-antigen derived peptides, nucleic acids encoding them, cancer antigen presenting cells and tumor cells. Taking a case comprising two types of substances for inducing a specific immune response to a cancer cell, as an example, a single type of cancer-antigen derived peptide and another type of cancer-antigen derived peptide may be contained; or a single type of cancer-antigen derived peptide and a single type of substance selected from a cancer-antigen protein, a nucleic acid encoding a cancer-antigen protein or a cancer-antigen derived peptide, a cancer antigen presenting cell and a tumor cell, may be contained.

A medicine according to the present invention can be used as a cancer vaccine therapy. In this case, a TLR agonist and LAG-3 protein, a variant or derivative thereof serve as adjuvants and enhance induction of an immune response by a “substance for inducing a specific immune response to a cancer cell”. As shown in Examples (later described), when the TLR agonist and LAG-3 protein, a variant or a derivative thereof are used in combination, an extremely high antitumor effect can be obtained even at a low dose at which no effect is obtained when they are used alone.

In the specification, the “adjuvant” refers to a molecule(s), which is administered together with a substance for inducing an immune response to enhance induction of the immune response.

A cancer vaccine therapy can be used for prevention or treatment of cancer. In the specification, prevention or treatment of cancer refers to causing at least one of phenomenon: a decrease in tumor size, delay or termination of increase in tumor size, inhibition (delay or termination) of cancer metastasis, inhibition (delay or termination) of proliferation of cancer cells, inhibition (delay or termination) of cancer recurrence, and mitigation of one or more of symptoms associated with cancer.

In the specification, the term “cancer” is used in the broadest sense thereof. Examples of the cancer include, but are not limited to, astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma, neurilemmoma, neurofibrosarcoma, neuroblastoma, pituitary tumor (for example, for pituitary adenoma), medulloblastoma, melanoma, brain tumor, prostate cancer, head and neck cancer, esophageal cancer, renal cancer, renal cell carcinoma, pancreatic cancer, breast cancer, lung cancer, colon cancer, colorectal cancer, stomach cancer, skin cancer, ovarian cancer, bladder cancer, fibrosarcoma, squamous cell carcinoma, neuroectodermal tumor, thyroid tumor, lymphoma, leukemia, multiple myeloma, hepatocellular carcinoma, mesothelioma and epidermoid carcinoma.

The present invention includes an adjuvant including a TLR agonist and LAG-3 protein, a variant or derivative thereof, for use in a cancer vaccine therapy. The TLR agonist is an adjuvant to be administered in combination with LAG-3 protein, a variant or derivative thereof; whereas LAG-3 protein, a variant or derivative thereof can be an adjuvant to be administered in combination with the TLR agonist. Such adjuvants may be administered to a patient together with various “substances inducing a specific immune response to a cancer cell”. The term “together” used herein does not mean concurrent administration but means that a TLR agonist and LAG-3 protein, a variant or derivative thereof are administered to a patient such that the TLR agonist and LAG-3 protein, a variant or derivative thereof can function as adjuvants in accordance with a cancer vaccine therapy, in vivo (of a patient's body) and ex vivo.

In the present invention, a medicine comprising a TLR agonist for combined administration with LAG-3 protein, a variant or derivative thereof may be provided; or a medicine comprising LAG-3 protein, a variant or derivative thereof for combined administration with a TLR agonist may be provided.

Also, in the present invention, a medicine comprising a TLR agonist for combined administration with a substance for inducing a specific immune response to a cancer cell and LAG-3 protein, a variant or derivative thereof may be provided. The medicine comprising a TLR agonist can act, with LAG-3 protein, a variant or derivative thereof as adjuvants, to enhance induction of immune response by a substance for inducing a specific immune response to a cancer cell, preferably, a cancer-antigen derived peptide. In the present invention, a medicine comprising LAG-3 protein, a variant or derivative thereof for combined administration with a substance for inducing a specific immune response to a cancer cell and a TLR agonist may be provided. The medicine comprising LAG-3 protein, a variant or derivative thereof can act, with a TLR agonist as adjuvants, to enhance induction of immune response by a substance for inducing a specific immune response to a cancer cell, preferably a cancer-antigen derived peptide.

In a medicine comprising a TLR agonist and LAG-3 protein, a variant or derivative thereof according to the present invention, the TLR agonist and LAG-3 protein, a variant or derivative thereof can be used as active ingredients of the medicine. The medicine comprising a TLR agonist and LAG-3 protein, a variant or derivative thereof as active ingredients according to the present invention, as shown in Examples (described later), can be used as an anti-cancer agent having a high antitumor effect. In the specification, the “anti-cancer agent” refers to an agent that can be used for preventing or treating cancer. The medicine serving as an anti-cancer agent may contain a TLR agonist and LAG-3 protein, a variant or derivative thereof as active ingredients, and may further contain a substance for inducing a specific immune response to a cancer cell.

In the present invention, a medicine serving as an anti-cancer agent contains a substance for inducing a specific immune response to a cancer cell, preferably, a cancer-antigen derived peptide, as an active ingredient, and may contain a TLR agonist and LAG-3 protein, a variant or derivative thereof as adjuvants.

Components of a medicine according to the present invention are dissolved in a water-soluble solvent to prepare pharmaceutically acceptable salts and a preparation containing the components in the salt form can be administered to patients. Examples of such pharmaceutically acceptable salts include physiologically acceptable water-soluble salts such as a sodium salt, a potassium salt, a magnesium salt and a calcium salt, which are adjusted to have physiological pH by a buffer. Other than water-soluble solvents, a nonaqueous solvent can be used. Examples of the nonaqueous solvent include alcohols such as ethanol and propylene glycol.

A medicine according to the present invention can be used as a pharmaceutical composition, which has any dosage form orally or parenterally administrated. The dosage form is not particularly limited. Examples of the dosage form of a pharmaceutical composition can include a liquid (for example, an injection), a dispersant, a suspension, a tablet, a pill, a powdered drug, a suppository, a powder, a fine grain, a granule, a capsule, syrup, a nasal drop and an ear drop.

A medicine according to the present invention, if it is used as a cancer vaccine, can be orally or parenterally administered. As parenteral administration, for example, intraperitoneal administration, subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration or intranasal administration can be employed.

A preparation of a medicine according to the present invention can be produced in accordance with a method known in the art. In the preparation of a medicine according to the present invention, pharmaceutically acceptable carriers and additives (such as an excipient, a binder, a dispersant, a disintegrant, a lubricant, a solvent, a solubilizer, a coloring agent, a flavoring agent, a stabilizer, an emulsifier, a suspending agent, an absorption promoter, a surfactant, a pH adjustor, a preservative and an antioxidant) may be used. Examples of the carriers and additives include pharmaceutically acceptable organic solvents such as water, saline, a phosphate buffer, dextrose, glycerol and ethanol, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, glutamic acid, aspartic acid, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, glucose, corn starch, microcrystalline cellulose, a surfactant, sodium bisulfite, sodium bisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, methyl paraben, phenylethyl alcohol, ammonia, dithiothreitol, beta mercaptoethanol, sodium carbonate, sodium borate, sodium phosphate, sodium acetate, sodium bicarbonate, sucrose, powdered sugar, sucrose, fructose, glucose, lactose, reduced malt sugar syrup, powdered reduced malt sugar syrup, glucose fructose syrup, fructose glucose sugar, honey, erythritol, aspartame, saccharin, saccharin sodium and gelatin.

When a medicine according to the present invention contains a peptide, an absorption enhancer for improving absorption of a poorly absorbable drug (hard-to-transmucosally absorbed drug), such as a peptide, may be used. Examples thereof include surfactants such as a polyoxyethylene lauryl ether, sodium lauryl sulfate and saponin; bile salts such as glycocholic acid, deoxycholic acid and taurocholic acid; chelating agents such as EDTA and salicyl bile salt; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid and mixed micelle; an enamine derivative, a N-acyl collagen peptide, a N-acyl amino acid, a cyclodextrin, a chitosan and a nitric oxide donor.

When a medicine according to the present invention contains a peptide, the medicine is encapsulated in e.g., polylactic acid/glycolic acid (PLGA) microcapsules or allowed to adsorb to porous hydroxyapatite fine particles to prepare a sustained release medicine, or the medicine is applied to a pulsed release iontophoresis patch system to prepare a transdermal absorbent.

A medicine according to the present invention may be a single preparation containing a TLR agonist and LAG-3 protein, a variant or derivative thereof or may be a combination of different preparations, i.e., a preparation containing a TLR agonist and a preparation containing LAG-3 protein, a variant or derivative thereof.

Also a medicine according to the present invention may be a single preparation containing a substance for inducing a specific immune response to a cancer cell, a TLR agonist and LAG-3 protein, a variant or derivative thereof or may be a combination of different preparations, i.e., a preparation containing a substance for inducing a specific immune response to a cancer cell, a preparation containing a TLR agonist, and a preparation containing LAG-3 protein, a variant or derivative thereto. More specifically, a preparation containing two types of components selected from a substance for inducing a specific immune response to a cancer cell, a TLR agonist and LAG-3 protein, a variant or derivative thereof may be used in combination with a preparation containing the other remaining component. As long as a combination of three types of components is provided, a preparation containing any two types of components and a preparation containing any two types of components, may be used in combination.

A medicine according to the present invention may be provided in kit form. In the case of a kit, the kit may contain a TLR agonist and LAG-3 protein, a variant or derivative thereof, or may contain a substance for inducing a specific immune response to a cancer cell, a TLR agonist and LAG-3 protein, a variant or derivative thereof.

A medicine according to the present invention, if it is used as a cancer vaccine, may contain additional adjuvants. Non-limiting examples of the additional adjuvants include sedimentary adjuvants such as aluminum hydroxide, sodium hydroxide, aluminum phosphate, calcium phosphate, alum and carboxyvinyl polymer, and oily adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, liquid paraffin, lanolin, montanide ISA763AV and montanide ISA51.

A medicine according to the present invention may be used in combination with other anti-cancer agents in any embodiment and may be used in combination with a radiation therapy and a surgical treatment. Examples of other anti-cancer agent include low molecular compounds such as adriamycin, daunomycin, mitomycin, cisplatin, vincristine, epirubicin, methotrexate, 5-fluorouracil, aclacinomycin, nitrogen mustard, cyclophosphamide, bleomycin, daunorubicin, doxorubicin, vincristine, vinblastine, vindesine, tamoxifen, and dexamethasone; and proteins such as cytokines activating immunocompetent cells (for example, human interleukin 2, human granulocyte macrophage colony stimulating factor, human macrophage colony stimulating factor, and human interleukin 12).

The present invention includes a method for treating cancer by administering a medicine according to the present invention in a therapeutically effective dose. The therapeutically effective dose can be appropriately determined by those skilled in the art depending upon e.g., the symptoms, age, sex, body weight and sensitivity difference of the patient, the administration method, the administration interval and type of preparation.

In the present invention, it is possible to treat or prevent a patient's disease by administering a TLR agonist and LAG-3 protein, a variant or derivative thereof to a patient in need thereof. In the present invention, a method for inducing a specific immune response to a cancer cell by administering a TLR agonist and LAG-3 protein, a variant or derivative thereof, to a patient in need thereof, is provided.

In treating or preventing a patient's disease or in a method for inducing a specific immune response to a cancer cell, a substance for inducing a specific immune response to a cancer cell, preferably, a cancer-antigen derived peptide, may be further administered.

A medicinal effect of a substance for inducing a specific immune response to a cancer cell, preferably a cancer-antigen derived peptide, can be more strongly exerted by inducing a specific immune response to a cancer cell.

A medicine according to the present invention is preferably used for combined administration.

The use of a medicine according to the present invention in combined administration means that a TLR agonist and LAG-3 protein, a variant or derivative thereof may be administered to a patient in any combination at the same time or different times. If a medicine according to the present invention contains a substance for inducing a specific immune response to a cancer cell, the substance for inducing a specific immune response to a cancer cell, a TLR agonist and LAG-3 protein, a variant or derivative thereof may be administered in any combination at the same time or different times.

In administering components at the same time, the components may be administered in single dosage form at the same time; more specifically, a TLR agonist and LAG-3 protein, a variant or derivative thereof may be mixed at the time of administration to prepare a dosing preparation and administered at the same time.

In administering components at different times, a TLR agonist is administered, and thereafter, LAG-3 protein, a variant or derivative thereof may be administered; or LAG-3 protein, a variant or derivative thereof is administered and thereafter a TLR agonist may be administered.

In administering components at the same time if a medicine according to the present invention contains a substance for inducing a specific immune response to a cancer cell, the components may be administered in single dosage form at the same time; more specifically, the substance for inducing a specific immune response to a cancer cell, a TLR agonist and LAG-3 protein, a variant or derivative thereof may be mixed at the time of administration to prepare a dosing preparation and administered at the same time.

In administering components at different times, the substance for inducing a specific immune response to a cancer cell is administered, and thereafter, a TLR agonist and LAG-3 protein, a variant or derivative thereof are administered at the same time or different times; or the TLR agonist and LAG-3 protein, a variant or derivative thereof may be administered at the same time or different times, and thereafter, the substance for inducing a specific immune response to a cancer cell may be administered. Alternatively, one of the TLR agonist and LAG-3 protein, a variant or derivative thereof is administered, and thereafter, the substance for inducing a specific immune response to a cancer cell is administered, and then, the other one of the TLR agonist and the LAG-3 protein, a variant or derivative thereof, may be administered. In administering the components at different times, the components may be administered based on the properties and the dose interval of individual components, in other words, based on the dosing regimens of the individual components.

The disclosures of all Patent Literatures and Non Patent Literatures cited in the specification are incorporated herein in their entirety by reference.

EXAMPLES

Now, the present invention will be described based on Example; however, the present invention is not limited to this. The present invention can be modified in various ways by those skilled in the art without departing from the scope of the present invention and such modification is included in the scope of the present invention.

1. Cancer Vaccine

In accordance with the protocol shown in FIG. 1, the effects of adjuvants in cancer vaccines using a cancer-antigen derived peptide were compared.

Material

As cancer model mice, DBA/2 mice, to which P815 cells (DBA/2mouse-derived mouse mastocytoma) were grafted, were put in use.

As a cancer-antigen derived peptide, a peptide (hereinafter referred to as “PIA peptide”) consisting of a partial sequence of PIA protein was used, which is a tumor antigen of P815 tumor and known to be restrictively presented by an MHC H-2Ld. The amino acid sequence of PIA peptide are represented by LPYLGWLVF (SEQ. ID No: 40).

As PIA CTL, a T cell expressing a T cell receptor and recognizing PIA peptide was used. In the experiment, the spleen was taken out from a PIA-CTL transgenic mouse that the present inventors possessed and a positive ratio was checked by TCR Vα 8.3 (TCR marker obtained by gene introduction). Based on the positive ratio, the number of PIA-CTL cells was determined and administered.

As the adjuvants, the following substances were used.

Whole pertussis bacterial cell (PT) (BioFarma, Bandung, Indonesia)

Poly I:C (TLR3 agonist) (Invivogen, SanDiego, USA)

CpG (TLR9 agonist) (Invivogen, SanDiego, USA)

IFA (incomplete Freund's adjuvant) (Seppic, Paris, France)

LAG-3Ig (Adipogen, SanDiego, USA)

Method

P815 tumor cells were subcutaneously inoculated to DBA/2 mice in an amount of 5×10⁵ cells per mouse. The day of inoculation was specified as Day 0. On Day 8, PIA CTL (2.5×10⁵ cells per mouse) were intravenously injected. On Day 9 and Day 16, an admixture of PIA peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneously injected. Mice were divided into 9 groups each consisting of 5 mice and the following adjuvants were used, respectively.

Group 1: PBS (Control)

Group 2: IFA (50 μL/mouse)

Group 3: PT (1×10⁸/mouse)

Group 4: Poly I:C (50 μg/mouse)

Group 5: Poly I:C (50 μg/mouse)+CpG (10 μg/mouse)

Group 6: LAG-3Ig (1 μg/mouse)

Group 7: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

Results

Changes in tumor size (mm³) of all mice on and after Day 7 are shown in FIGS. 2 to 8.

In all of Groups 1 to 6, tumor sizes gradually increased and mice mostly died in the middle. The number of mice survived until Day 70 without increasing tumor size was 0 in Groups 2, 3 and 6, 1 in Groups 1 and 4, and 3 in Group 5; however, in Group 7, an increase in tumor size was not observed in all 5 mice and the mice survived until Day 115.

2. Infiltration of Immune Cells into Cancer Tissue

Cancer model mice were prepared by subcutaneously inoculating P815 tumor cells (5×10⁵ cells per mouse) to DBA/2 mice. The day of inoculation was specified as Day 0. On Day 8, PIA CTL (2.5×10⁵ cells per mouse) were intravenously injected. Day 9 and Day 14, an admixture of PIA peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneously injected. The following adjuvants were used in respective mouse groups.

Group 1: IFA (50 μL/mouse)

Group 2: Poly I:C (50 μg/mouse)

Group 3: LAG-3Ig (1 μg/mouse)

Group 4: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

On Day 21, a tumor tissue was taken and slides of tumor tissue sections were prepared. Thereafter, a tissue image was observed by staining with hematoxylin-eosin; at the same time, cell nuclei and immune cells (CD4 cells and CD8 cells) were stained with a fluorescent dye.

The following reagents were used in the fluorescent tissue staining.

Cell nucleus: ProLongR Gold Antifade Reagent with DAPI (Invitrogen)

CD4 cell primary antibody: Rat Anti-Mouse CD4 Purified IgG2b. Clone: GK1.5 (eBioscience)

CD4 cell secondary antibody: Mouse monoclonal (2B 10A8) Anti-Rat IgG2b heavy chain (Alexa FluorR 647), (abcam)

CD8 cell primary antibody: Rat Anti-Mouse CD8a Purified IgG2a. Clone: 53-6.7 (eBioscience)

CD8 cell secondary antibody: Mouse monoclonal (2A 8F4) Anti-Rat IgG2a heavy chain (Alexa FluorR 488) (abcam)

Tissue images stained with hematoxylin-eosin are shown in FIG. 9A and images of cell nucleus and immune cells stained with a fluorescent dye are shown in FIG. 9B. When LAG-3Ig and poly I:C were used in combination as an adjuvant, significant infiltration of CD4 cells and CD8 cells into a tumor tissue was observed.

3. Maintenance of Ability to Reject the Same Tumor in Mice Once Rejected a Tumor

P815 tumor cells were subcutaneously inoculated to DBA/2 mice in an amount of 5×10⁵ cells per mouse and used as cancer model mice. The day of inoculation was specified as Day 0. On Day 8, P1A CTL (2.5×10⁵ cells per mouse) were intravenously injected. On Day 9 and Day 14, an admixture of P1A peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneously injected. As an adjuvant, LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse) was used.

Subsequently, to DBA/2 mice caused no increase in tumor size even if P815 tumor cells were inoculated, and determined as rejecting a tumor, and naive DBA/2 mice to which no treatment was applied, again P815 tumor cells (1×10⁶ cells per mouse) or L1210 tumor cells (1×10⁶ cells per mouse) were subcutaneously inoculated. To DBA/2 mice determined as rejecting a tumor, the tumor cells were again inoculated on Day 115 after the first inoculation of P815 tumor cells.

FIG. 10 (left) shows changes in average tumor size (mm³) of mice after inoculation of L1210 cells; whereas, FIG. 10 (right) shows changes in average tumor size (mm³) of mice after inoculation of P815 cells. In both figures, open circle represents DBA/2 mice determined as a tumor rejection mice, and closed circle represents naive DBA/2 mice.

In naive DBA/2 mice, an increase in tumor size was observed even if either one of P815 cells and L1210 cells were inoculated. In contrast, in mice determined as rejecting a tumor growth of P815 cells, an increase in tumor size by inoculation of another type of tumor cells, i.e., L1210 cells, was observed; however, an increase in tumor size by P815 cell inoculation was not observed. It was confirmed that an ability to reject the same tumor is maintained.

4. Determination of the Proliferative Ability of Immune Cells and Measurement of the Cytokines in Cell Supernatant

P815 tumor cells were subcutaneously inoculated to DBA/2 mice in an amount of 5×10⁵ cells per mouse to prepare cancer model mice. The day of inoculation was specified as Day 0. On Day 8, PIA CTL (2.5×10⁵ cells per mouse) were intravenously injected. On Day 9 and Day 14, an admixture of PIA peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneously injected. The following adjuvants were used in respective mice groups.

Group 1: IFA (50 μL/mouse)

Group 2: Poly I:C (50 μg/mouse)

Group 3: LAG-3Ig (1 μg/mouse)

Group 4: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

Of the lymph nodes in the axilla and inguinal region, the lymph node close to a tumor site was taken on Day 21 and immune cells were separated. The immune cells (1.5×10⁵ cells) and P815 cells (4×10⁴ cells) irradiated with 100 Gy were co-cultured for 3 days.

Immune cell proliferative ability was determined by adding ³H-thymidine (37 KBq/well) to the culture supernatant and measuring the radioactivity of ³H-thymidine taken in the cells, 4 hours later.

The amounts of cytokines in the cell supernatant were measured by using Bio-Plex Pro mouse cytokine 23-Plex Immunoassay kit (BIO-RAD).

Measurement results of immune cell proliferative ability are shown in FIG. 11A and measurement results of cytokines are shown in FIG. 11B.

In a case of using a combination of LAG-3Ig and poly I:C as an adjuvant, immune cell proliferative ability increased. Among the cytokines, IFN-γ, GM-CSF, IL-4, IL-5 and IL-17A were found to increase in production when LAG-3Ig and poly I:C were used in combination as an adjuvant.

5. Measurement of Cell-Surface Marker Molecules of Immune Cells

P815 tumor cells were subcutaneously inoculated to DBA/2 mice in an amount of 5×10⁵ cells per mouse to prepare cancer model mice. The day of inoculation was specified as Day 0. On Day 8, PIA CTL (2.5×10⁵ cells per mouse) were intravenously injected. On Day 9 and Day 14, an admixture of PIA peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneously injected. The following adjuvants were used in respective mice groups.

Group 1: IFA (50 μL/mouse)

Group 2: Poly I:C (50 μg/mouse)

Group 3: LAG-3Ig (1 μg/mouse)

Group 4: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

Of the lymph nodes in the axilla and inguinal region, the lymph node close to a tumor site was taken on Day 21, CD8 and Vα 8.3 expression positive cell group (killer T cells) or CD4 and Vα 8.3 expression positive cell group (helper-T cells) were collected.

Expression levels of cell-surface marker molecules (on CD4 cells and CD8 cells), i.e., PD-1, BTLA, TIGIT and LAG-3, were measured. As antibodies against the cell surface marker molecules, the following molecules were used.

PD-1: Anti-mouse CD279 (PD-1) PE. Clone: J43 (eBioscience)

BTLA: Anti-mouse CD272 (BTLA) PE. Clone: 8F4 (eBioscience)

TIGIT: PE anti-mouse TIGIT (Vstm3) Antibody. Clone: 1G9 (BioLegend)

LAG-3: Anti-mouse CD223 (Lag-3) PE. Clone: eBioC9B7W (C9B7W) (eBioscience)

The results of CD8 cells are shown in FIG. 12A and the results of CD4 cells are shown in FIG. 12B.

When a combination of LAG-3Ig and poly I:C was used as an adjuvant, significant decreases in expression level of PD-1, TIGIT and LAG-3 were observed both in CD4 positive cells and CD8 positive cells; however, the decrease in expression level of BTLA was low.

6. Measurement of Cytokines Using B16-F10 Melanoma Inoculation Model Mice

To C57BL/6 mice, B16-F10 melanoma cells (1×10³ cells per mouse) were subcutaneously inoculated to prepare cancer model mice. The day of inoculation was specified as Day 0. On Day 8, an admixture of gp100 peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneously injected. The following adjuvants were used in respective mice groups. The amino acid sequence of gp100 peptide is represented by KVPRNQDWL (SEQ. ID No: 41).

Group 1: IFA (50 μL/mouse)

Group 2: Poly I:C (50 μg/mouse)

Group 3: LAG-3Ig (1 μg/mouse) Group 4: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

Of the lymph nodes in the axilla or inguinal region, the lymph node close to a tumor site (or both lymph nodes) was taken on Day 14 and immune cells were separated. The immune cells (3×10⁵ cells per well) were cultured in the presence of gp100 peptide (10, 5, 2.5, or 0 μg/mL).

Immune cell proliferative ability was determined by adding ³H-thymidine (37 KBq/well) to the culture supernatant and measuring the radioactivity of ³H-thymidine incorporated in the cells in the final 10 hours of the culture period of 3 days.

The IFN-γ production amount in the cell supernatant obtained by culturing 3 days in the presence of gp100 peptide (10 μg/m) was measured by Bio-Plex Pro mice cytokine 23-Plex Immunoassay kit (BIO-RAD).

Measurement results of immune cell proliferative ability are shown in FIG. 13A and measurement results of IFN-γ production amount are shown in FIG. 13B.

In FIG. 13A, open circle represents the results of Group 1, closed square Group 2, open square Group 3 and closed circle Group 4.

Even if B16-F10 melanoma inoculation model mice were used, if a combination of LAG-3Ig and poly I:C was used as an adjuvant, an increase of immune cell proliferative ability specific to gp100 tumor antigen and an increase of IFN-γ production were observed.

It was confirmed that when a combination of LAG-3Ig and poly I:C was used as an adjuvant, a significant activation effect on the immune system can be exerted regardless of the tumor model system and the type of peptide serving as an immune antigen.

7. Suppression Effect of Tumor Growth by Immunoadjuvant Combination

The following substances were used as adjuvants.

Poly I:C (TLR3 agonist) (Invivogen, SanDiego, USA)

RIBOXXOL (TLR3 agonist) (Riboxx, Radebeul, Germany)

MPL (TLR4 agonist) (Invivogen, SanDiego, USA)

Imiquimod (TLR7/8 agonist) (Invivogen, SanDiego, USA)

CpG (TLR9 agonist) (Invivogen, SanDiego, USA)

LAG-3Ig (Adipogen, SanDiego, USA)

P815 tumor cells were subcutaneously inoculated to DBA/2 mice in an amount of 5×10⁵ cells per mouse to prepare cancer model mice. The day of inoculation was specified as Day 0. On Day 7, PIA CTL (2.5×10⁵ cells per mouse) were intravenously injected. Day 8 and Day 15, an admixture of PIA peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneously injected. Mice were divided into 9 groups each consisting of 4 or 5 mice and the following adjuvants were used respectively.

Group 1: PIA peptide alone (control)

Group 2: Poly I:C (50 μg/mouse)

Group 3: Poly I:C (50 μg/mouse)+LAG-3Ig (1 μg/mouse)

Group 4: No PIA peptide, Poly I:C (50 μg/mouse)+LAG-3Ig (1 μg/mouse) alone

Group 5: RIBOXXOL (100 μg/mouse) Group 6: RIBOXXOL (100 μg/mouse)+LAG-3Ig (1 μg/mouse)

Group 7: MPL (10 μg/mouse)+LAG-3Ig (1 μg/mouse) Group 8: Imiquimod (50 μg/mouse)+LAG-3Ig (1 μg/mouse)

Group 9: CpG (10 μg/mouse)+LAG-3Ig (1 μg/mouse)

Change in tumor size (mm³) with respect to all mice on and after Day 7 are shown in FIGS. 14 to 22.

In Group 1, 5 out of 5 mice all died up to Day 40. Four out of 5 mice in Group 2, 5 out of 5 mice in Group 3, 3 out of 5 mice in Group 6, 2 out of 5 mice in Group 4 and 5 and 1 out of 5 mice in Groups 7 to 9 survived. In addition, 4 out of 5 mice in Group 3 and 3 out of 5 mice in Group 6 survived up to Day 66. A combination effect of adjuvants was demonstrated by comparing between Groups 2 and 3 and Groups 5 and 6.

8. Tumor Growth Inhibitory Effect by Combination of Immuno-Adjuvants in B16-F10 Melanoma Inoculation Model Mice

To C57BL/6 mice, B16-F10 melanoma cells (1×10⁵ cells per mouse) were subcutaneously inoculated to prepare cancer model mice. The day of inoculation was specified as Day 0. On Day 5 and Day 12, twice in total, an admixture of gp100 peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneously injected. The following adjuvants were used in respective mice groups.

Group 1: IFA (50 μL/mouse)

Group 2: Poly I:C (50 μg/mouse)

Group 3: LAG-3Ig (1 μg/mouse)

Group 4: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

Changes in tumor size (mm³) of all mice on and after Day 5 are shown in FIGS. 23 to 26.

Five mice all died on Day 31 in Group 1, on Day 35 in Group 2 and on Day 37 in Group 3. In contrast, five mice died in Group 4 on Day57. Compared to Group 1, a significant effect of extending survival time was not observed in Groups 2 and 3; however, the survival time of Group 4 was significantly extended compared to any one of Groups 1, 2 and 3.

9. Measurement of Vaccine Specific Immune Response Using Patient Blood Treated with Vaccine

To esophageal cancer patients having an HLA molecule of Type HLA-A*02:07/24:02 and hepatocellular carcinoma patients having an HLA molecule of Type HLA-A*24:02/26:01, a mixed solution of 2 mg of an HSP70 peptide (YGAAVQAAI: SEQ. ID No: 7), 2 mg of a GPC3 peptide (MVNELFDSL: SEQ. ID No: 16), 1 mg of IMP321 (LAG-3Ig) and 1.4 mg of “Hiltonol” (product name of Poly ICLC) in saline, was divided into four portions and each subcutaneously inoculated at 4 sites in the proximity of the limb groin area once a week in the first 2 months and once two weeks in the following month. After administration 10 times in total, 50 mL of the peripheral blood was sampled.

From the peripheral blood, PBMCs (peripheral blood mononuclear cells) were separated by using Ficoll-Paque Plus density gradient solution (GE Healthcare Bio-sciences), and the PBMCs were seeded in AIM-V+FBS culture medium in a 24-well plate at a ratio of 1×10⁶ cells per well and cultured.

On Day 1, Day 4, Day 8 and Day 12, 100 units/mL of recombinant IL2 (rIL2; Novartis) was added. On Day 0 and Day 7, 10 μg/mL of HSP70 peptide or GPC3 peptide, or HIV peptide (RYLRDQQLL: SEQ. ID No: 42) as a negative control or EBV peptide (TYGPVFMCL: SEQ. ID No: 43) as a positive control were added, and culture of the PBMCs was continued. On Day 14, CD8+ T cells were separated and recovered by negative selection using MACS beads.

ELISPOT assay was carried using the human IFN-γ ELISPOT PLUS kit (Mabtech). Using a 12-well plate previously coated with an anti-IFN-γ antibody, CD8+T responder cells (1×10⁴ cells/well) were cultured together with tumor cells (2×10⁴ cells/well) as a stimulator at 37° C. for 48 hours.

After an anti-IFN-γ antibody (7-B6-1) biotinylated was added as a secondary antibody and culture was carried out for 2 hours, an HRP (Horse Raddish Peroxidase) reagent was added and further staining was carried out by use of a TMB (tetramethylbenzidine) reagent.

The spots stained were automatically counted by use of the ImmunoSPOT S4 (Cellular Technology Ltd.).

The results are shown in FIG. 27A and FIG. 27B.

After an HSP70 peptide and a GPC3 peptide were administered 10 times in total, the number of spots associated with IFN-γ production advantageously increases compared to the negative control. It can be confirmed that a vaccine-specific immune response occurs.

Further, to hepatocellular carcinoma patients having an HLA molecule of Type HLA-A*24:02/24:02, colorectal cancer patients having an HLA molecule of Type HLA-A*02:01/11:01 and esophageal cancer patients having an HLA molecule of Type HLA-A*02:06/26:01 before vaccination, a mixed solution of 2 mg of an HSP70 peptide (YGAAVQAAI: SEQ. ID No: 42), 2 mg of a GPC3 peptide (MVNELFDSL: SEQ. ID No: 43), 1 mg of IMP321 (LAG-3Ig) and 1.4 mg of “Hiltonol” (product name of Poly ICLC) in saline was divided into four portions and each subcutaneously inoculated at 4 sites in the proximity of the limb groin area for a month, two months and two months at intervals of a week (administered 4 times, 8 times and 8 times) and 50 mL of the peripheral blood was sampled.

The peripheral blood samples each were separated and cultured in the same manner as the peripheral blood samples from the above esophageal cancer patients and the hepatocellular carcinoma patients, and stained with the same reagent. The stained spots were automatically counted in the same manner as above.

The results are shown in FIG. 28A, FIG. 28B and FIG. 28C.

After an HSP70 peptide and a GPC3 peptide were administered 4 times or 8 times, the number of spots associated with IFN-γ production advantageously increases compared to the negative control. It can be confirmed that a vaccine-specific immune response occurs.

Sequence Listing Free Text

SEQ. ID Nos: 1 to 15 represent the amino acid sequences of HSP70-derived peptides as a cancer-antigen derived peptide.

SEQ. ID Nos: 16 to 26 represent the amino acid sequences of GPC3-derived peptides as a cancer-antigen derived peptide.

SEQ. ID Nos: 27 to 39 represent the amino acid sequences of MUC1 derived peptides as a cancer-antigen derived peptide.

SEQ. ID No: 40 represents the amino acid sequence of PIA peptide.

SEQ. ID No: 41 represents the amino acid sequence of gp100 peptide.

SEQ. ID No: 42 represents the amino acid sequence of HIV peptide.

SEQ. ID No: 43 represents the amino acid sequence of EBV peptide.

SEQUENCE LISTING 

1. A medicine for treating a cancer in a patient, comprising a Toll-like receptor agonist, lymphocyte activation gene 3 (LAG-3) protein or a fusion protein of an extracellular domain of LAG-3 protein and IgG, and an HSP70-derived peptide consisting of SEQ ID NO: 7 and a GPC3-derived peptide consisting of SEQ ID NO:
 16. 2. The medicine according to claim 1, for combined administration of the Toll-like receptor agonist and LAG-3 protein or a fusion protein of the extracellular domain of LAG-3 protein and IgG, and an HSP70-derived peptide consisting of SEQ ID NO: 7 and a GPC3-derived peptide consisting of SEQ ID NO:
 16. 3. The medicine according to claim 1, for use in a cancer vaccine therapy.
 4. The medicine according to claim 1, wherein the Toll-like receptor agonist is a Toll-like receptor 3 agonist or a Toll-like receptor 9 agonist.
 5. The medicine according to claim 1, wherein the Toll-like receptor agonist is Poly I:C or a salt thereof.
 6. The medicine according to claim 5, wherein the Poly I: C is Poly ICLC.
 7. A medicine for inducing an immune response to a cancer cell in a patient, comprising a Toll-like receptor agonist, lymphocyte activation gene 3 (LAG-3) protein or a fusion protein of an extracellular domain of LAG-3 protein and IgG, and an HSP70-derived peptide consisting of SEQ ID NO: 7 and a GPC3-derived peptide consisting of SEQ ID NO:
 16. 